The Type-it Mutation Detect PCR Kit is especially designed to enable fast and reliable detection of mutations such as deletions, insertions, and translocations. The kit is based on highly specific HotStarTaq Plus DNA Polymerase and a patented buffer system, both of which enable reliable amplification of mutant loci by multiplex PCR without optimization. Subsequent analysis is straightforward and easy and can be carried out on agarose gels, automated electrophoresis instruments, and also by high-resolution capillary sequencing.

The Type-it Mutation Detect PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Sensitive detection of a mutated cancer-related gene.

The indicated amounts of DNA extracted from a lymphoma-related cell line (Ramos) were spiked into human leukocyte DNA, as indicated in the figure, and the mutated Ramos target was detected along with 2 internal controls. Using the Type-it Mutation Detect Kit, the mutated 357 bp gene was detected even when only 25 pg of DNA (4 cells) was present. The lymphoma-relevant mutation could not be detected when using preoptimized conditions (Mg2+ and polymerase) with a hot-start DNA polymerase from Supplier I — even when using 25 ng of DNA (4000 cells). Electrophoresis was performed on a 1.3% agarose gel. M: 100 bp ladder.

Superior preamplification of SNPs.

Two different PCR systems (12-plex and 5-plex), both including the SNP of interest, were amplified using the standard protocol for the Type-it Mutation Detect PCR Kit (Q) or using preoptimized conditions with a hot-start DNA polymerase from Supplier A. With Type-it Mutation Detect Kits, all fragments of interest were amplified without any optimization of PCR parameters and could successfully be used as input for the SNaPshot PCR system. Even after optimization of Mg2+ concentration to 4 mM and varying the polymerase concentrations, amplification using the kit from Supplier A resulted in missing fragments. Electrophoresis was performed on the QIAxcel System. The first and the last bands on each lane are Upper Alignment Marker and Lower Alignment Marker, respectively. M: Alignment marker.

Performance

The Type-it Mutation Detect PCR Kit outperformed kits tested from other suppliers and ensures reliable analysis of mutations. The kit is specifically developed and functionally validated for multiplex PCR-based analysis of mutations such as deletions or insertions and detection of genetically modified organisms or microbes (see figure "Sensitive detection of a mutated cancer-related gene"). The kit is also suitable for use as a preamplification method for commercially available SNP detection PCR-based systems such as the SNaPshot system provided by Applied Biosystems (see figure "Superior preamplification of SNPs").

Principle

The Type-it Mutation Detect PCR Kit is based on proprietary QIAGEN Multiplex Technology and is provided in a ready-to-use master mix format. The master mix contains optimized concentrations of HotStarTaq Plus DNA Polymerase, MgCl2, and dNTPs, and an innovative PCR buffer, specially developed for multiplex PCR-based detection of mutations or for preamplification of genomic SNP loci. It also includes the novel additive Factor MP, as well as a balanced combination of salts and additives. Together, these components enable comparable efficiencies for annealing and extension of all primers in the reaction (see figure "Stable and efficient annealing"), providing reliable high-yield multiplex amplification of all fragments in parallel. The kit includes dedicated protocols for the detection of mutations, as well as step-by-step advice on template amounts, cycle numbers, and PCR conditions and instrument details for different downstream analysis platforms such as agarose gels, capillary sequencers, the Agilent Bioanalyzer, and the QIAxcel Advanced System.

The combination of all components provided in the master mix and the dedicated protocol result in highly specific amplification of all fragments in parallel. Subsequent analysis is straightforward and easy and can be carried out on agarose gels, automated electrophoresis instruments, and also by high-resolution capillary sequencing.

Type-it Mutation Detect PCR Buffer

The Type-it Mutation Detect PCR Buffer ensures comparable amplification efficiency for all amplicons in a high-plex grade multiplex experiment and allows more mutation targets to be combined without the loss of amplification efficiency for any of the targets. In contrast to conventional PCR reagents, the Type-it Mutation Detect PCR Buffer contains a specially developed, balanced combination of salts and additives to ensure comparable efficiencies for annealing and extension of all primers in the reaction. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer-annealing conditions over a wider range of temperatures and Mg2+ concentrations than conventional PCR buffers. The need to optimize PCR by varying the annealing temperature or the Mg2+ concentration is dramatically reduced, or often not required. Commonly employed optimization procedures for multiplex PCR are virtually eliminated. The buffer also contains the synthetic Factor MP (see figure "Stable and efficient annealing"), which allows efficient primer annealing and extension, irrespective of primer sequence. Factor MP increases the local concentration of primers at the DNA template and stabilizes specifically bound primers.

Q-Solution

The Type-it Mutation Detect PCR Kit is supplied with Q-Solution. This innovative PCR additive facilitates amplification of difficult templates by modifying the melting behavior of DNA. Use of this unique reagent will often enable or improve suboptimal PCR. Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer-template system and is not toxic.

CoralLoad Dye

The Type-it Mutation Detect PCR Kit is supplied with CoralLoad Dye (see figure "CoralLoad Dye"), which contains a gel-loading reagent and two gel-tracking dyes that improve pipetting visibility and facilitate estimation of DNA migration distance, as well as optimization of agarose gel run time. When using CoralLoad Dye, in the multiplex PCR reaction, the amplicons can be directly loaded onto an agarose gel or the QIAxcel Advanced System without prior addition of loading buffer. CoralLoad dyes do not interfere with most downstream enzymatic applications. However, for reproducible results, purification of PCR products prior to enzymatic manipulation is recommended.

If using capillary sequencers for detection, CoralLoad Dye must not be used.

Procedure

The Type-it Mutation Detect PCR Kit includes dedicated, application-specific protocols, optimized for simultaneous amplification and subsequent detection of loci carrying mutations or SNPs to ensure reliable results for routine analysis or establishment of new assays. Reactions can be set up at room temperature, ensuring greater convenience and ease of use.

Fast and simple way to reliable genotyping results

The Type-it Mutation Detect PCR Kit enables fast and easy multiplex assay development for accurate and reproducible genotyping results. Whether its detection of translocations, deletions, insertions, or preamplification of genomic loci for SNP analysis methods such as the SNaPshot (Applied Biosystems), just add the template and primers, and start the thermal cycler program according to the optimized protocol. The reaction mixture contains all of the reagents required for mutation-specific multiplex PCR and enables accurate results without the need for lengthy optimization procedures, when compared with current methods (see figure "Successful multiplex PCR-based genotypic analysis").

Time and cost savings due to straightforward procedure

Some studies require analysis of a large number of different mutations of a certain gene related to a disease (e.g., deletions, translocations, or insertions) requiring a large number of PCR reactions when performing singleplex or low-plex grade PCR analysis, thereby leading to increases in both costs and analysis time. The Type-it Mutation Detect PCR Kit ensures comparable amplification efficiency for all amplicons in a high-plex grade multiplex experiment and allows more mutation targets to be combined ensuring significant time and cost savings (see figure Sensitive detection of a mutated cancer-related gene).

Reliable preamplification of SNPs

The Type-it Mutation Detect PCR Kit is also highly suited for multiplex PCR-based preamplification of SNPs (e.g., the SNaPshot system from Applied Biosystems). The kit outperformed kits tested from other suppliers and consistently delivers reliable results, without the need for tedious optimization procedures (see figure "Superior preamplification of SNPs").

Applications

The Type-it Mutation Detect PCR Kit is used to analyze mutations, deletions, insertions, duplications, translocations, and for SNP preamplification (e.g., SNaPshjot Multiplex Kit) and is used in diverse research fields such as: